Project 3 Summary Abstract The deep and durable anti-tumor responses that are possible with tumor infiltrating lymphocyte (TIL) and chimeric antigen receptor-expressing (CAR) T cell therapy illustrate the potential of T cells as a therapeutic platform for cancer. Effective T cell therapy depends upon rigorous clonal expansion following engagement of the antigen-specific T cells. Cytokines play an essential role in promoting this clonal expansion of T cells. Infusion of recombinant human IL-2 (rhIL2, Proleukin) enhances adoptive T cell therapy by enhancing the proliferation of T cells post-transfer, but this comes at the cost of significant dose-limiting toxicity. Preconditioning of patients with lymphodepleting chemotherapy or radiation also improves engraftment and expansion of adoptively transferred T cells, in part by elevating the concentrations of important cytokines like IL-7 and IL-15 that regulate homeostatic T cell proliferation. However, preconditioning is also associated with appreciable toxicity related to the non-specific effects of chemotherapy and radiation. Improved methods of promoting specific expansion and persistence of engineered T cells following adoptive transfer with reduced toxicity are therefore needed. This proposal takes advantage of the orthogonal cytokine - cytokine receptor platform developed by the Garcia Lab at Stanford. This proposal will combine this novel orthogonal cytokine system with CAR T cell therapies developed within the Milone lab at Penn to overcome the need for lymphodepletion and improve the potency of engineered T therapies using approaches that can be readily translated into a clinical setting. This includes exploring the use of oncolytic adenovirus to deliver orthogonal cytokine within the tumor microenvironment to promote CAR T cell expansion (Aim 1), engineering T cells with autocrine secretion of orthogonal cytokine to circumvent the need for endogenous IL-2, improving proliferation of CAR T cells while depriving regulatory T cells of IL-2 that mediate adaptive resistance (Aim 2) and the development of a novel orthogonal IL-21 receptor with enhanced activation of STAT3 that is predicted to have important effects on T cell metabolism, survival and differentiation that are distinct from IL-2 and beneficial for tumor-specific immunity (Aim 3).